Our research focuses on the biochemical events involved in apoptosis. Apoptosis is an important mode of cell death relevant for many developmental, immunologic, and pathologic processes. Unraveling the signaling events for diverse apoptotic stimuli should lead to a better understanding of diseases in which cell death is dysregulated (cancer, AIDS, degenerative disorders) as well as the normal regulation of immune responses. Most models for apoptosis suggest that various stimuli, with unique cellular targets, induce a common cell death signal that triggers the controlled destruction of the cell. These later events appear to involve the activation of ICE-family proteases (caspases). Recent work has examined the role of Bcl-2 proteins and caspases in the regulation of apoptosis. We developed a transient transfection death assay so that the effect of various gene products on apoptosis can be readily assessed. Overexpression of Bcl-2 family members in the mouse T cell hybridoma 2B4 efficiently blocked killing induced by cytotoxic agents but not anti-Fas whereas CrmA, an ICE-like caspase inhibitor, showed the opposite pattern. These results suggested that there are distinct biochemical pathways leading to apoptosis with differential sensitivity to Bcl-2 and caspase inhibitors. However, since Bcl-2 has been reported to inhibit Fas-mediated cytotoxicity in some transfected tumor lines, it was important to determine whether Bcl-2 showed stimulus-dependent activity in normal T cells. We have extended our findings to normal cells by transiently transfecting T cell blasts. Once again, overexpression of Bcl-2, Bcl-xL, or adenovirus E1B 19K blocked apoptosis induced by cytotoxic agents but not anti-Fas. CrmA preferentially inhibited Fas-mediated apoptosis while Ac-DEVD-CHO blocked both signaling paths. These data strongly support two apoptotic signaling pathways in T cells; cytotoxic agents use a Bcl-2-sensitive, CrmA-resistant CPP32-like caspase path, while Fas-induced apoptosis use a Bcl-2-resistant, CrmA-sensitive ICE-like caspase and CPP32-like caspase pathway. Since normal T cells express Bcl-2 family members following activation, the lack of interference with Fas-mediated cytotoxicity may be necessary to allow the elimination of self-reactive T cells and the proper regulation of immune responses. Other collaborative research projects have examined mechanisms of CTL-mediated cytotoxicity and the characterization of recombinant Bcl-2 protein.